Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently emerged pathogen. To predict and control the evolutionary and pathogenic potential of SARS-CoV, a better understanding of the basic biology and pathogenesis should lead to the development of vaccines and therapies. We have developed a full length infectious cDNA for mouse hepatitis virus (MHV), transmissible gastroenteritis virus (TGEV) and,SARS-CoV. The overall hypothesis in this proposal is that deletion or rearrangement of selected ORFs and/or "rewiring " of the coronavirus transcription network will identify virulence alleles, attenuate SARS-CoV pathogenesis in vivo, stabilize against recombination and reversion, and result in candidate vaccines that protect against in vivo challenge. Attenuated viruses developed during the course of these studies will also serve as seed stocks for safe, killed vaccines. In aim 1, Dr. Baric tests that hypothesis that the SARS-CoV group specific genes encode luxury functions in vitro. Two SARS-CoV group specific ORFs encode IFN antagonists that likely influence pathogenesis (see Palese, Project 3) and viable viruses have been isolated that contain deletions in these ORFs. We will delete systematically knockout the SARS-CoV group specific ORFs and characterize the phenotype of rescued viruses. The goal is to isolate replication competent viruses that lack group specific ORFs, are attenuated and protect against wildtype challenge in vivo. In aim 2, Dr. Baric tests the hypothesis that the SARS-CoV gene order rearranged viruses recombine less frequently than wildtype viruses and are attenuated in animal models. In aim 3, Dr. Baric tests the hypothesis that the transcriptional regulatory sequences of SARS-CoV can be rewired and that these mutants will be highly resistant to recombination with wildtype and other coronaviruses. The goal is to create a genome platform that can 1) house a suite of attenuated alleles and 2) contain genetic traps that impart lethal genome instabilities, triggered after recombining with other coronaviruses. In aim 4, the in vivo pathogenesis of these live attenuated and select killed viruses are compared in mice and ferrets. We will also determine if select mutants induce immune responses that protect against wildtype challenge. The project simultaneously increases our understanding of the molecular biology of SARS-CoV replication and transcription, the molecular mechanisms governing coronavirus recombination and strategies to impede RNA recombination repair of candidate vaccine strains, and the evolutionary potential of the SARS-CoV genome as an emerging human pathogen.